Research in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Intracellular protein degradation plays a critical part in controlling the levels of cellular regulatory proteins and is an essential element of protein quality control systems. Protein degradation within the cytosol is carried out by ATP-dependent proteases, which are multimeric complexes made up of three essential components: a recognition domain that interacts with specific signals in target proteins, an ATP-driven protein unfoldase that structurally disrupts the bound protein and translocates it to the third component, and a tightly associated self-compartmentalized protease. Our research encompasses structural and biochemical analysis of the ATP-dependent Clp and Lon proteases from bacteria and from human mitochondria and assays of their biological activities. Studies are focused on four major areas: the basis for substrate selection by ClpA, ClpX, and Lon;structural dynamics of ClpP and the mechanism by which unfolded proteins enter the degradation chamber;conformational changes in the AAA+ domains of Clps and Lon that contribute to their activities;and the role of human ClpXP in mitochondrial function and signaling under conditions of stress. One universal mechanism of protein recognition operates by controlled exposure of a subset of amino acids at the N-terminus of proteins (N-degrons). Binding of N-degrons by components of the degradative machinery (N-recognins) allows these proteins to be targeted for degradation by ATP-dependent proteases. In bacterial cells ClpAP degrades proteins with N-degrons, and the adaptor protein, ClpS, is the N-recognin that binds to N-degrons. We found that delivery of substrates occurs with only one molecule of ClpS per ClpA hexamer despite the presence of 6 equivalent N-domains capable of binding ClpS. Limiting the number of substrates trying to enter the narrow axial channels of ClpA avoids steric clashes between them. ClpS has a bipartite binding mode in which the globular domain interacts with a ClpA N-domain and the N-terminal 20 amino acids interact with the axial channel. The first molecule of ClpS binds to ClpA with high affinity and blocks binding of subsequent ClpS molecules by sterically excluding other ClpS molecules. To learn how proteins with N-degrons arise in E. coli, we have begun to identify all the proteins that acquire N-degrons. We used a ClpS affinity column to capture proteins with exposed N-degrons from cell extract and have identified unique proteins bearing N-degrons. We found that the number of proteins captured from ClpS or ClpA mutant cells is 20-50 times greater than that obtained from wild-type cell extracts, suggesting the proteins are substrates for degradation by ClpAP/ClpS. During the next phase, we will isolate proteins accumulating when other components of the system, such as the Phe-aminotransferase, are mutated and will begin to determine the regulatory and physiological effects of targeting the substrates that have been identified. Studies with ClpP have been focused on the structural changes that are needed to allow substrate entry into the degradation chamber. Cryo electron microscopy shows that the axial pore of ClpP expands to a diameter of greater than 18 when ClpA binds. To investigate the conformation of the ClpP N-terminal loops during this structural rearrangement, we engaged in a collaboration to get the crystal structure of ClpP in the open-channel state. Acyldepsipeptide antibiotics (ADEPs) induce an open-channel conformation of ClpP that can take up unfolded proteins and is highly activated for peptide degradation. The crystal structure of ADEP bound to ClpP showed that ADEP binds to the hydrophobic groove on the surface of ClpP that serves as the docking site for the IGF/L loops of ClpA and ClpX. A short peptide with the sequence IGF was modeled in the position of the bound ADEP and confirmed that the ADEP-bound state mimics the state activated by ClpA or ClpX binding. By wedging between the subunits in the heptameric ring, ADEP causes the subunits to rotate upward and outward, which allows the N-terminal loops to snap into a parallel array expanding the axial channel and removing side chains from the path translocating substrates use. Deletion of the N-terminal loops allows ClpP to degrade unfolded proteins but destabilizes the interactions between the heptameric rings. We propose that the orientation of subunits affects both crowding of the N-terminal loops and the handle regions that form the interface between heptamers. Binding of ClpX and ClpA (or ADEP) causes the subunits to rotate, opening the axial channel and reorienting the handle regions to favor interaction between the rings. Because ClpA and ClpX stabilize an open configuration of the ClpP N-terminal loops, there is no additional energy requirement for translocating substrates into ClpP once substrates are unfolded and extruded through the axial channels of ClpA or ClpX. A breakthrough has been obtained in the structural analysis of Lon proteases. Lons are composed of complexes of one type of subunit containing a tandem alignment of the recognition, chaperone/unfoldase, and protease domains. In a collaborative study, the crystal structure of nucleotide-bound hexameric state of Lon protease was solved. The structure confirmed that Lon protease domains are oriented facing the internal chamber of the chaperone domains, creating a large chamber in which unfolded or partially unfolded proteins are directly exposed to the proteolytic active sites. The structure also reveals that Lon subunits are in alternating conformational states around the ring. The closed state with tightly bound ADP cannot be adjacent to another subunit in the same state, indicating that the subunits within the hexamer function in a sequential rather than concerted manner. Lon has a unique gating mechanism in which loops protruding from the chaperone domain, which are subject to nucleotide-dependent reorganization, combine with either N-domains or inserted membrane-spanning domains to form a gated passageway for recognizing and screening appropriate substrates. This structure opens the way to detailed analysis of conformational states and functional residues in Lon proteases that underlie the important biological roles of this enzyme, which has been implicated in a range of vital processes. Human ClpX and ClpP function within the mitochondrial matrix and are needed for mitochondrial integrity and for cell survival. Depletion of hClpP or hClpX following treatment with siRNA also leads to cell death. Down regulation of hClpP affects the timing and extent of apoptotic cell death in response to DNA damage, death receptor binding, and kinase inhibition. The similarity in response to 3 divergent stress signaling pathways suggests that hClpP alters the basal structure or physiology of mitochondria. Down regulation of hClpP sensitizes cells to various drugs that have been used to treat cancer and partially reverse the drug resistance of multidrug resistant cells. We have begun to investigate the changes in the mitochondrial proteome in response to depletion and overexpression of hClpP and HClpX. Analysis of 2D gels followed by mass spectrometry has identified more than 30 proteins whose levels increased within 16 hours of depletion of hClpP. A number of the proteins identified are involved in the response to oxidative and other stress. To identify low abundance substrates, future efforts will be directed at improving the sensitivity of the mass spectroscopic methods used and particular focus will be placed on enriching for potential physiological targets by trapping substrates in inactive forms of ClpP and by pull-down procedures using ClpX complexes.